1. Field of the Invention
The present invention relates generally to the preparation of lipid vesicles, or liposomes, and particularly to the preparation of such vesicles having components, which may be membrane-incorporated or encapsulated, to render such vesicles suitable for therapeutic or diagnostic applications.
2. Brief Description of the Prior Art
Lipid vesicles, whether single- or multi-compartmented, have walls composed of lipids, particularly lipid mixtures including at least one phospholipid, in the form of continuous membranes. For a general view of the preparation, properties and uses of lipid vesicles, reference is made to Papahadjopoulos et al., (Eds.), Liposomes, Ann. N.Y. Acad. Sci., Vol. 308 (1978); Tom et al (Eds.), Liposomes and Immunobiology, Elsevier North Holland Inc., N.Y. (1980); Gregoriadis et al, (Eds.) Liposomes in Biological Systems, John Wiley & Sons, N.Y. (1980); Knight (Ed.), Liposomes: From Physical Structure to Therapeutic Applications, Elsevier North Holland Inc., N.Y. (1981); and Gregoriadis (Ed.), Liposome Technology, Vol. 1, CRC Press, Boca Raton, Fla. (1984). As discussed in these references, vesicles have been prepared in the past by numerous methods, each of which has certain advantages and disadvantages.
The earliest of these methods employs a film-formation techniques which involves the preparation of multilamellar vesicles (MLVs) by depositing the membrane components, dissolved in suitable organic solvents, as a coating film on the internal wall of a glass vessel, e.g., by evaporation. An aqueous solution of the materials to be encapsulated is introduced into the vessel as a hydrating mixture. The vessel is shaken or rotated for a period of time, to peel individual layers of the lipid coating to form vesicles which encapsulate or entrap the hydration mixture. The size of the resulting vesicles can vary from a fraction of a micron to several microns. Long hydration periods (10-20 hours) are normally required for satisfactory entrapment. The degree of entrapment depends upon such physical and mechanical factors as the nature of the surface upon which the lipid coating is deposited, the manner of agitation, the thickness of the deposited coating, etc. Also, the resulting vesicles can vary widely in size, for example, ranging between 0.1 microns to several microns. As the number of lipid vesicles produced is a function of the effective surface of the vessel, scale-up to achieve production-size quantities of lipid vesicles by this method would require very large vessels.
Alternatively, small unilamellar vesicles (SUVs) have been prepared by sonication of lipid mixtures or MLVs prepared by the film-formation technique described above. Typical sizes of SUVs are usually in the range of 20-100 nm and the size distribution is usually narrower than for the MVLs. While SUVs are useful for encapsulating materials of low molecular weights, e.g., drugs, they are too small to efficiently encapsulate proteins such as enzymes or antibodies, nucleic acids and other high molecular weight polymers. For that purpose, the SUVs can be enlarged to form large unilamellar vesicles (LUVs) by a series of freezing and thawing cycles in the hydrating medium and in the presence of alkali metal ions. This method is even more time-consuming than the film-formation technique described above, in that the additional steps of sonication and freezing and thawing are required.
Another method for forming vesicles employs a reverse-phase evaporation technique. In this method, lipids are dissolved in an appropriate organic solvent or solvent mixture having the same density as the hydrating mixture. The lipid solution is intimately dispersed in the hydrating mixture by sonication or vigorous shaking leading to formation of an emulsion. The organic solvent is subsequently evaporated to a level whereat reverse micelles are formed. Further evaporation and shaking of the remaining solution results in the formation of LUVs. The disadvantages of this method are the technical difficulty associated with the emulsification process and the risk of denaturing sensitive molecules, such as proteins and nucleic acids, during the emulsification process due to their prolonged contact with organic solvents.
Also, in infusion method, which is similar to the reverse-phase evaporation technique, has been employed, whereby lipids are initially dissolved in an organic solvent, e.g., ether, ethanol, etc. The resulting solution is injected as a tiny stream into the warm hydrating mixture, to allow the solvent to dissolve or evaporate. As a result, lipids are dispersed in the hydrating mixture and form vesicles. Vesicles formed either by ethanol or ether infusion are relatively small (0.4 microns or less) and unsuitable for applications requiring a large ratio of entrapped volume to membrane surface, e.g., immunodiagnostics. Vesicles formed by ethanol infusion exhibit relatively poor encapsulation efficiency as compared to those produced by ether infusion. Vacuum or heat may be applied to accelerate solvent evaporation. The danger does exist, however, that sensitive molecules may be denatured by the heat or by contact with the organic solvent.
A further method employs a detergent-removal technique. In such method, lipids are introduced into an aqueous medium containing a detergent, which solubilizes them. The detergent is subsequently removed by exhaustive dialysis whereupon the lipids become insoluble in the agueous medium and tend to form vesicles. Long dialysis times are required to completely remove the detergent. Even very small amounts of detergent remaining in the medium will affect the ultimate permeability of the vesicle and, hence, its usefulness as a diagnostic or therapeutic reagent.
When considering a method for the commercial production of vesicles for therapeutic or diagnostic applications, many requirements exist. The method of choice should be fast, easy to scale-up for production quantities, and maximize the encapsulation or entrapment of solutes present in the hydrating mixture within the vesicle. Also, the method should not adversely affect the chemical stability of the components involved in the vesicle formation process.
The prior art methods, described above, each suffers from one or more disadvantages. Usually, the combined time for preparation and purification of the vesicles is of the order of many hours. For scale-up purposes, a method that depends on liposome formation at the surface of a container, such as in the film-formation technique or the detergent removal technique, is likely to be difficult to scale-up. The surface area of a spherical container increases as the square of the diameter, whereas the volume increases as the cube of such diameter. Eventually, large and cumbersome apparatus would be required for commercial production. Several of the methods mentioned above result in the production of vesicles having small size and poor encapsulation efficiency. Methods of vesicle formation in which conditions, such as contact with organic solvents, emulsification, heat, etc., that may cause denaturation of biological molecules are not preferred.